Mixed Solid-State Fermentation of Okara and Copra Meal by Probiotics with Non-Starch Polysaccharide Enzymes and Its Effects on the Growth Performance and Ileal Microbiota in Broilers
, Shih-Yao Lin 1, Yu-Hsiang Yu 1,* and Yeong-Hsiang Cheng 1,*
Round 1
Reviewer 1 Report
Thank you for invitation of peer review. Sorry, I missed the due date. Below, I will report on the review of submitted paper. I think that this manuscript is lack of novelty and should more show and discuss physiological data. In the publication of the journal, I judged that more drastic correction is necessary. Thank you for your consideration.
The manuscript describes effect of FOCM supplementation on the growth performance and ileal microbiota in broiler. Various experimental techniques are examined, but as a paper, there is a lack of discussion and explanation.
First, an interesting reaction has been observed at FOCM 0.125, but a rationale should be given as to why such a concentration was set in this case.
Secondly, the experimental methods should be shown precisely and detailed information on HPLC and ELISA should be given. And as for the analysis of fatty acids, how were they measured by HPLC?
Third, what were the blood properties measured for in this case? I do not understand the intention.
Fourth, why did such a change occur on day 21, when even on day 7 we can see the results changing? This point needs to be discussed.
Fifth, why did feed intake and feed requirement change? In other words, why did the availability of nutrients change, and a discussion of the mechanism in this regard is needed.
Sixth, the way the statistics in the table are marked with significance symbols is not correct.
Author Response
Comments and Suggestions for Authors:
Thank you for invitation of peer review. Sorry, I missed the due date. Below, I will report on the review of submitted paper. I think that this manuscript is lack of novelty and should more show and discuss physiological data. In the publication of the journal, I judged that more drastic correction is necessary. Thank you for your consideration.
Response:
Thank you for your helpful comments. We have revised our manuscript accordingly and feel that your comments helped clarify and improve our manuscript. Please find our response (in red) to reviewer’s specific comments below.
- The manuscript describes effect of FOCM supplementation on the growth performance and ileal microbiota in broiler. Various experimental techniques are examined, but as a paper, there is a lack of discussion and explanation.
Answer:
Thank you for your comments. We have added more explanation and information about the broiler growth performance and ileal microbiota as well the correlation of them in the discussion section, as follows:
In Discussion section:
Page 15, line 509-537.
“4.3. Animal study
4.3.1. Effects of FOCM on the growth performance of broiler
Since fermentation is a pre-digestion process, macromolecular proteins are first decomposed into peptides or amino acids and improve the nutritional quality to help young animals increase nutrient utilization [59]. The FOCM produced in this study not only had a large amount of amino acid but also contained a large amount of lactic acid bacteria. It is an important condition because the supplementation of lactic acid bacteria in the feed will be helpful in improving the growth performance of broilers due to its lactic acid products [60]. As lactic acid bacteria were enriched in FOCM, it could significantly improve the feed efficiency of broilers without affecting their body weight gain and at the same time could save the feed costs (Table 3). Since okara and CM are fermented by microorganisms, a large amount of protein is decomposed into amino acids, and lactic acid fermentation produces a sour flavor. In addition to lactic acid, this study also showed an increase in butyric acid (Figure 3c). A previous study revealed that lactic acid (along with other organic acids: fumaric acid and citric acid) could decrease the gastric pH, thus, it could indirectly eliminate the proliferation of acid-sensitive bacteria. Likewise, butyric acid (and other organic acids: formic acid, acetic acid, propionic acid, and sorbic acid) could reduce the pH in the GIT by directly acting upon the cell wall of Gram-negative bacteria [61, 62]. Moreover, specifically in butyric acid, another study has demonstrated that this butyric acid could increase the digestion and nutrition absorption of broilers [63, 64], which aims to improve broiler growth performance. In addition, the energy usage of different groups may also affect feed intake. The FOCM which was produced in this study contained high amounts of fiber, and the feed metabolizable energy was lower than that of the control group. It is indicated that the addition of FOCM will help increase the appetite of broilers. Accordingly, along with the lower energy value will make broilers consume more feed effectively represented by higher feed intake and lower the FCR but no reducing in body weight on FOCM1.25 group. This was different from the FOCM5.0 group where the FCR content was close to the CTRL group. Since the NSP content is closely related to the ability of the poultry digestive tract to absorb feed nutrients [21], we assumed that the FOCM5.0 group had higher NSP content due to having more fermented okara and CM.”
Moreover, we also provided in detail the correlation between FCR (as growth performance indicator) and microbiota analysis
Page 18, line 624-643.
“The FCR, which is defined as the ratio between feed inputs and product outputs during the rearing period, is extensively used to determine feed efficiency (yield per unit feed) [83, 84]. In chickens, the FCR is calculated as a ratio of feed eaten to body weight gained. As a result, chickens with a low FCR require less feed per kilogram gained and are the most efficient at converting feed to mass [85]. In broilers, the genus Lactobacillus has been shown to be linked to FCR [86, 87]. In addition, the presence of Lactobacillus caused a low FCR in chickens [85]. In the current study, the FOCM2.5 group has the most abundant Lactobacillus (64.84%, Table 8) amongst the groups, and the FCR at day 35 and the whole experiment period has the lowest FCR (Table 3).
Escherichia coli and Shigella, species of Enterobacter, are known to overgrow in the gut to cause damage to the intestinal mucosa, erosion of villi, and intestinal cells, thereby reducing the ability of animals to absorb nutrients [88-90]. Therefore, the reduced relative abundance of Escherichia-Shigella was possible due to the lessened damage of intestinal villi (day 21) in the diet supplemented with FOCM. As for its effect on growth performance, a previous study revealed that the reduced Escherichia coli in the intestine could help improve the growth performance of broilers [87]. In addition, it would be even better if the reduced Escherichia coli was accompanied by an increased Lactobacillus [87, 91]. Previous studies indicated that the fecal microbial composition was related to the growth performance of broilers [80, 92, 93].”
- First, an interesting reaction has been observed at FOCM 0.125, but a rationale should be given as to why such a concentration was set in this case.
Answer:
Thank you very much for your constructive comments. In order to make the points raised in this result, we have added some information to explain this in discussion section.
In Discussion section:
Page 16, line 528-537.
“4.3. Animal study
4.3.1. Effects of FOCM on the growth performance of broiler
The FOCM which was produced in this study contained high amounts of fiber, and the feed metabolizable energy was lower than that of the control group. It is indicated that the addition of FOCM will help increase the appetite of broilers. Accordingly, along with the lower energy value will make broilers consume more feed effectively represented by higher feed intake and lower FCR but no reduction in body weight in the FOCM1.25 group. This was different from the FOCM5.0 group where the FCR content was close to the CTRL group. Since the NSP content is closely related to the ability of the poultry digestive tract to absorb feed nutrients [21], we assumed that the FOCM5.0 group had higher NSP content due to having more fermented okara and CM.”
Page 18, line 618-620.
“4.3. Animal study
4.3.4. Bacterial richness analysis of chicken ileum
Similarly, according to the FCR result, the FOCM1.25 group was significantly lower than control group, and the relative abundance of Firmicutes was significantly higher (p < 0.05).”
Page 18, line 624-630.
“4.3. Animal study
4.3.4. Bacterial richness analysis of chicken ileum
The FCR, which is defined as the ratio between feed inputs and product outputs during the rearing period, is extensively used to determine feed efficiency (yield per unit feed) [83, 84]. In chickens, the FCR is calculated as a ratio of feed eaten to body weight gained. As a result, chickens with a low FCR require less feed per kilogram gained and are the most efficient at converting feed to mass [85].”
- Secondly, the experimental methods should be shown precisely and detailed information on HPLC and ELISA should be given. And as for the analysis of fatty acids, how were they measured by HPLC?
Answer:
Thank you for your comment. We have added the methods as follow:
In Methods section:
Page 5, line 213-217.
“2.7. Blood chemistry and antibody titer analysis
The detection method of ND titer antibody used the hemagglutinin inhibition (HI) test; as it has been noted, the HI test is a highly accurate measure of a herd's immunological health and resistance to ND, particularly for evaluating the protective response to ND vaccination [49]. The IB titer antibody was detected by enzyme-linked immunosorbent assay (ELISA) kit (BioChek, Gouda, The Netherlands).”
In addition, we also added the analysis of lactic acid and butyric acid content methods as follow:
Page 4, line 160-171.
“2.4. Determination of moisture, neutral detergent fiber, residual reducing sugars, lactic acid and butyric acid content
The content of lactic acid and butyric acid was determined by high-performance liquid chromatography (HPLC). The FOCM was incubated at 50°C in a water bath and ultrasonically shaken for 20 minutes. The homogenate was mixed in the water and then filtered with a 0.22 μm filter membrane. The extract was then analyzed for lactic acid and butyric acid content using HPLC and compared with standard. Lactic acid (Sigma-Aldrich) and butyric acid (Sigma-Aldrich) standards were prepared and analyzed a minimum of three times. By using the C18 column, 5 μm inner diameter 4.6 mm x 25 cm long, the lactic acid mobile phase was 0.01 mol/L phosphoric acid solution and the butyric acid mobile phase was 80% acetonitrile and 0.02% phosphoric acid. HPLC analysis of lactic acid and butyric acid was performed at the same flow rate of 1.0 mL/min and wavelengths of 210 nm and 206 nm. The concentration of lactic acid and butyric acid were determined based on the slope of the standard curve.”
- Third, what were the blood properties measured for in this case? I do not understand the intention.
Answer:
Thank you for your comment. We have explained more about blood properties in discussion section, as follows:
In Discussion section:
Page 16-17, line 539-589.
“4.3.2. Effects of FOCM on blood biochemistry and serum antibody titers of broilers
During the experiment, the FOCM5.0 group produced higher amounts of albumin than other groups (Table 4). The total protein in serum is composed of albumin and globulin, wherein the globulin includes immunoglobulin such as IgA. Hence, feeding lactic acid bacteria can ameliorate the integrity of the intestinal barrier by upregulating the tight junction-related genes (Zone occludens-1 (ZO-1) and occludin) and increasing the production of Ig-A in Peyer’s patch [65]. In addition, these lactic acid bacteria are needed for the mucosa to compete with pathogens (toxins, viruses, and bacteria) for the binding site on the intestinal epithelial cell surface [66]. Moreover, IgA is needed for the mucosa to prevent the attachment of antigens (toxins, viruses, and bacteria) by direct neutralization [67]. In a similar study in pigs, the addition of LAB to swine's diet has more benefits; it can not only inhibit pathogens in the gastrointestinal tract but also improve the microbial balance in the intestine, as well as improve the function of the intestinal barrier and up-regulate intestinal mucosal immunity [66]. Celi [68] showed that the intestinal mucosal immune system is made up of a system of lymphoid tissues called the gut-associated lymphoid tissue (GALT). In the GALT, lymphocytes can be found in three separate places: (1) the Peyer’s patches, which are lymphoid follicles (T and B lymphocytes, mainly B-cells in adults) located in the mucosa and extending to the submucosa in the ileum; (2) the lamina propria lymphocytes, which are mainly IgA secretory B lymphocytes; and (3) the intra-epithelial lymphocytes, which are located in the epithelium, at the basolateral spaces between the epithelial cells, beneath the tight junctions. Other cells, like M-cells, are in the intestinal epithelium. The role of M-cells is to endocytose antigens and transport them to antigen-presenting cells (APCs; macrophages and dendritic cells), which are located in the underlying tissue, for further digesting the antigen and presenting it to T-cells [68].
Chickens have maternal immunity against ND and IB viruses which begin to decrease in titer at 5-10 days old [69, 70]. Therefore, our study carried out ND and IB vaccination with active vaccines at 4- days of age and revaccination at 14- days of age. Furthermore, antibody titers were investigated when the broilers were 21 and 35 days old. At the time of vaccination, antibody titers have not yet appeared, so it takes time to detect them. In general, antibody titers can be detected at least 14 days after vaccination [69]. Previous studies have shown that natural products (natural resin), probiotics, and fermented products as feed additives induce antibody responses in vaccinated chickens against ND and IB viruses [71-75]. However, to the best of our knowledge, there have been no studies revealing the response of the combination of probiotics and fermented products produced by the SSF method. The supplementation of probiotics as a feed additive to chickens vaccinated against ND can increase the antibody response to the ND virus but has no significant difference in antibody titers between the treatment and control groups [72], and the result of this study was similar to our current study (Table 5). On the other hand, feed supplemented with fermented yeast given to chickens showed an increase in antibody titer of the ND vaccine against the ND virus by investigating the availability of mannan-binding lectin (MBL). In addition, Kjærup [76] also investigated MBL in IB-vaccinated chickens. Accordingly, compared with previous studies, it is necessary to investigate the immune response between vaccinated and unvaccinated chickens (or before and after vaccination) and do some tests to check MBL specifically. The MBL is a plasma protein involved in innate immune defense against various microorganisms, including viruses. The MBL binds to mannose and N-acetylglucosamine oligosaccharides and their derivatives presented on the surface of microorganisms [75] and plays an important role in humans and chickens to protect the host from viral infection [76]. We assumed that by offering probiotics and fermented products orally, with the short chicken rearing period of broilers and the formation of antibody response that takes several weeks from vaccination until antibody titer measurements can be observed, it will certainly cause local (not systemic yet) effects.”
- Fourth, why did such a change occur on day 21, when even on day 7 we can see the results changing? This point needs to be discussed.
Answer:
Thank you for your comment. Since we vaccinated the chicks when they were 4 days old. However, before the end of the experiment (35 days of age, In Methods section: page 4-5, line 195-200), we analyzed the growth-related characteristics of the birds every 7 days (in Result section: page 9 line 335 and 339, Tables 2 and 3), but the blood-related analysis (in Result section: page 10, line 358 and 364, Tables 4 and 5) were analyzed only at 21 and 35 days of age.
We then describe more information about this in Discussion section, as follows:
in Discussion section:
Page 17, line 562-567.
“4.3.2. Effects of FOCM on blood biochemistry and serum antibody titers of broilers
Chickens have maternal immunity against ND and IB viruses which begin to de-crease in titer at 5-10 days old [69, 70]. Therefore, our study carried out ND and IB vaccination with active vaccines at 4-days of age and revaccination at 14-days of age. Furthermore, antibody titers were investigated when the broilers were 21 and 35 days old. At the time of vaccination, antibody titers have not yet appeared, therefore, we detected ND and IB antibodies after day 21.”
- Fifth, why did feed intake and feed requirement change? In other words, why did the availability of nutrients change, and a discussion of the mechanism in this regard is needed.
Answer:
Thank you for your constructive comments. We have added information in detail to describe its mechanism, as follows:
In Discussion section:
Page 15-16, line 509-537.
“4.3. Animal study
4.3.1. Effects of FOCM on the growth performance of broiler
Since fermentation is a pre-digestion process, macromolecular proteins are first decomposed into peptides or amino acids and improve the nutritional quality to help young animals increase nutrient utilization [59]. The FOCM produced in this study not only had a large amount of amino acid but also contained a large amount of lactic acid bacteria. It is an important condition because the supplementation of lactic acid bacteria in the feed will be helpful in improving the growth performance of broilers due to its lactic acid products [60]. When the lactic acid bacteria were combined with FOCM, it could significantly improve the feed efficiency of broilers without affecting their body weight gain and at the same time could save the feed costs (Table 3). Since okara and CM are fermented by microorganisms, a large amount of protein is decomposed into amino acids, and lactic acid fermentation produces a sour flavor. In addition to lactic acid, this study also showed an increase in butyric acid (Figure 3c). A previous study revealed that lactic acid (along with other organic acids: fumaric acid and citric acid) could decrease the gastric pH, so, it could indirectly eliminate the proliferation of acid-sensitive bacteria. Likewise, butyric ac-id (and other organic acids: formic acid, acetic acid, propionic acid, and sorbic acid) could reduce the pH in the GIT by directly acting upon the cell wall of Gram-negative bacteria [61, 62]. Moreover, specifically in butyric acid, another study has demonstrated that this butyric acid could increase the digestion and nutrition absorption of broilers [63, 64], which aims to improve broiler growth performance. In addition, the energy usage of different groups may also affect feed intake. The FOCM which was produced in this study contained high amounts of fiber, and the feed metabolizable energy was lower than that of the control group. It is indicated that the addition of FOCM will help increase the appetite of broilers. This, along with the lower energy value will make broilers consume more feed effectively represented by higher feed intake and lower the FCR but no reducing in body weight on FOCM1.25 group. This was different from the FOCM5.0 group where the FCR content was close to the CTRL group. Since the NSP content is closely related to the ability of the poultry digestive tract to absorb feed nutrients [21], we assumed that the FOCM5.0 group had higher NSP content due to having more fermented okara and CM.”
- Sixth, the way the statistics in the table are marked with significance symbols is not correct.
Answer:
Thank you for your suggestion. We have revised it as follows:
In Result Section:
Page 8, line 318.
“Figure 3. Effect of 55% initial moisture of fermentation okara and CM with the combination of mixed probiotics and NSPases supplementation on reducing sugar content (a); lactic acid contents (b); and butyric acid contents (c) with different fermentation duration.”
Page 14, line 437.
On Table 8, specifically about the relative abundance of Phylum Firmicutes.
|
CTRL1 |
FOCM1.25 |
FOCM2.5 |
FOCM5.0 |
　p-value |
|||||
|
relative abundance (%) |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
|
|
Phylum |
|||||||||
|
Firmicutes |
52.87 b |
5.74 |
96.66a |
2.05 |
85.41a |
18.84 |
92.70a |
3.30 |
0.02 |
|
Proteobacteria |
10.93 |
6.00 |
0.45 |
0.34 |
2.15 |
2.28 |
1.67 |
1.35 |
0.06 |
|
Bacteroidetes |
14.32 |
2.39 |
0.66 |
0.44 |
8.06 |
11.73 |
4.86 |
4.18 |
0.14 |
|
Genus |
|||||||||
|
Bifidobacterium |
8.23 |
6.43 |
0.01 |
0.01 |
1.42 |
2.42 |
1.06 |
1.69 |
0.08 |
|
Lactobacillus |
3.86 |
2.66 |
59.30 |
25.57 |
64.84 |
37.41 |
62.31 |
32.65 |
0.08 |
|
Lachnospiraceae_unclassified |
5.15 |
2.68 |
0.03 |
0.02 |
1.78 |
2.94 |
0.74 |
0.94 |
0.06 |
|
Escherichia-Shigella |
2.26a |
0.19 |
0.31b |
0.07 |
1.01b |
0.80 |
0.50b |
0.43 |
0.01 |
Author Response File: 
Author Response.pdf
Reviewer 2 Report
English in the introduction and results part is not very good. There are numerous typos and awkward expressions. I included some of them at the end of this report but I did not check throughout the manuscript. The insufficient background information to explain the application of fermented feed, incomplete discussion of the scientific and practical implications of the results make the submission unsuitable for publication. There was much missing information to explain the results and their applicability.
Comments that lead to major revision of the manuscript as follow:
1. Introduction
There are many kinds of probiotics, and many probiotics have been applied in feed fermentation. In this study, L. acidophilus, L. delbrueckii, and L. salivarius and C. butyricum were used as probiotics. More information about these bacteria, such as their probiotic function and application, should be given to explained why these bacteria are selected. moreover, there are many researches on probiotic fermented feed. The introduction lacks the application of fermented feed and the application status of okara and copra in feed.
2. Experimental design
This study only compared the growth of broilers without FOCM and with FOCM feed. The difference in the growth performance and ileal microbiota may be due to the different feed formula. FOCM was prepared by fermentation. It is not clear whether fermentation improves the nutritional value of okara and copra. Therefore, two controls are required for this study. A group of control was fed normal diet , and the other group was fed with unfermented okara and copra.
Fermentation will affect many nutrients, including protein, amino acids, polysaccharides, cellulose, etc. This paper introduced okara and copra meal contains unusable non-starch polysaccharides (NSP) fiber. therefore, this research studied the fermentation conditions of exogenous non-starch polysaccharide degrading enzymes (NSPases) and probiotics for the mixed okara and CM. In this study, only evaluate the effect of solid-state fermentation on the reducing sugar content, lactic acid contents, butyric acid contents and soy globulins contents of okara and CM. However, lactic acid contents, butyric acid contents and soy globulins contents is not related to non-starch polysaccharides. Therefore, this paper should study the change of indexes which are related to non-starch polysaccharides, such as fiber content, polysaccharide content, monosaccharide content, etc.
3. Figure
The title of figures can be simplified. For example, the title of Figure 1 can be changed to: Effect of different initial moisture content on Lactobacillus species (a)and C. butyricum (b) growth in SSF of okara and CM.
Figure 2: It is really confusing about the group of probiotics, NSPases and mix. There is no introduction to these three groups in the material method. In addition, Lactobacillus species and Clostridium butyricum were used as probiotics in this study. Why only Lactobacillus was detected, but not Clostridium butyricum.
Author Response
Comments and Suggestions for Authors:
English in the introduction and results part is not very good. There are numerous typos and awkward expressions. I included some of them at the end of this report but I did not check throughout the manuscript.
Response:
Thank you for your helpful comments. We have revised our manuscript accordingly and feel that your comments helped clarify and improve our manuscript. Please find our response (in red) to reviewer’s specific comments below.
The insufficient background information to explain the application of fermented feed, incomplete discussion of the scientific and practical implications of the results make the submission unsuitable for publication. There was much missing information to explain the results and their applicability.
Answer:
Thank you for your suggestion. We have rewritten the introduction section to explain I detail about the fermented feed and its practical application.
Comments that lead to major revision of the manuscript as follow:
- Introduction
There are many kinds of probiotics, and many probiotics have been applied in feed fermentation. In this study, L. acidophilus, L. delbrueckii, and L. salivarius and C. butyricum were used as probiotics. More information about these bacteria, such as their probiotic function and application, should be given to explained why these bacteria are selected. moreover, there are many researches on probiotic fermented feed. The introduction lacks the application of fermented feed and the application status of okara and copra in feed.
Answer:
Thank you for your comment. In introduction, we described in detail about these bacteria as probiotics, as follows:
In Introduction section:
Page 2-3, line 91-103.
Lactobacillus species (Lactobacillus acidophilus, Lactobacillus delbrueckii, and Lactobacillus salivarius) [35, 36] and Clostridium butyricum [37] are known to ferment okara and improve the nutritional status and growth performance of animals. Specifically, Lactobacillus species are capable of producing bacteriocin and short-chain fatty acids (lactate and butyrate), which help lower pH and maintain acidity in the animal gut [38]. Such bacteria are bile resistant because they have bile salt hydrolase (BSH) enzyme that help hydrolyze bound bile and reduce its toxic effects [39]. Although there are no reports of using Clostridium butyricum alone to ferment soybean dregs, Clostridium butyricum is commonly used in soybean meal fermentation along with Lactobacillus [40-42]. Compared to other probiotics, Clostridium butyricum is heat-resistant and can be stored in dry form at room temperature through the pelleting process of feed products [43]. In addition, Clostridium butyricum is resistant to low pH and high bile concentrations; hence, it can reach and colonize the small intestine after ingestion in broilers.”
Also, we have added more information about the application of fermented feed, as follows:
In Introduction section:
Page 3, line 104-108.
“Previous studies have investigated that SSF can improve the nutritional quality of animal feed by increasing nutrient bioavailability and reducing anti-nutritional factors in single feed ingredients [44], such as palm kernel cake [45], and rapeseed meal [46]. The present study was conducted to investigate the ability of SSF in mixed ingredients, okara, and CM.”
- Experimental design
This study only compared the growth of broilers without FOCM and with FOCM feed. The difference in the growth performance and ileal microbiota may be due to the different feed formula. FOCM was prepared by fermentation. It is not clear whether fermentation improves the nutritional value of okara and copra. Therefore, two controls are required for this study. A group of control was fed normal diet, and the other group was fed with unfermented okara and copra.
Answer:
Thank you for your constructive comment, we have revealed that SSF improved the nutritional value of okara and CM by investigating the amino acid content (Table 1, data not shown in the manuscript). As shown below (Table 1), there were 15 amino acids that increased after 60 h of fermentation (p < 0.05).
Table 1. amino acid analysis of mixed okara and CM before and after soild-state fermentation.
|
Items |
0 h |
60 h |
　 p-value |
||
|
Mean |
SD |
Mean |
SD |
||
|
asp |
1.97 |
0.05 |
2.38 |
0.04 |
0.0003 |
|
thr |
0.58 |
0.02 |
0.74 |
0.03 |
0.002 |
|
ser |
0.70 |
0.03 |
0.88 |
0.06 |
0.0103 |
|
glu |
3.06 |
0.09 |
3.71 |
0.07 |
0.0005 |
|
pro |
0.72 |
0.06 |
0.78 |
0.21 |
0.6522 |
|
gly |
0.77 |
0.02 |
0.97 |
0.02 |
0.0002 |
|
ala |
0.83 |
0.01 |
1.03 |
0.02 |
<.0001 |
|
cys |
0.17 |
0.02 |
0.22 |
0.02 |
0.0423 |
|
val |
0.92 |
0.02 |
1.12 |
0.02 |
0.0001 |
|
met |
0.24 |
0.03 |
0.28 |
0.05 |
0.2791 |
|
ile |
0.68 |
0.02 |
0.83 |
0.02 |
0.0002 |
|
leu |
1.26 |
0.04 |
1.54 |
0.03 |
0.0005 |
|
tyr |
0.33 |
0.03 |
0.44 |
0.04 |
0.0165 |
|
phe |
0.77 |
0.02 |
0.94 |
0.02 |
0.0003 |
|
lys |
0.81 |
0.03 |
1.04 |
0.03 |
0.0008 |
|
his |
0.37 |
0.01 |
0.46 |
0.02 |
0.0012 |
|
arg |
1.57 |
0.06 |
1.86 |
0.06 |
0.0035 |
Furthermore, according to the information stated in Introduction section (page 2, line 55-60 and line 65-66), okara and CM contain high non-starch polysaccharides (NSP) fiber and the intact NSP present in the poultry digestive tract are known to inhibit the digestive process and interfere with intestinal balance, leading to problems associated with wet dropping (wet feces) and even intestinal diseases. Hence, we did not include the unfermented okara and CM.
In Introduction section:
Page 2, line 55-60.
“In addition, the carbohydrates contained in okara are considered to be generally un-available non-starch polysaccharides (NSP) fiber, which are mainly composed of man-nan, galactomannan, and cellulose [18-20]; intact NSP present in the poultry digestive tract is known to inhibit the digestive process and interfere with intestinal balance, leading to problems associated with wet dropping (wet feces) and even intestinal diseases [21].
In Introduction section:
Page 2, line 65-66.
Similar to okara, CM contains high water content and harbors indigestible NSP [19].
- Fermentation will affect many nutrients, including protein, amino acids, polysaccharides, cellulose, etc. This paper introduced okara and copra meal contains unusable non-starch polysaccharides (NSP) fiber. therefore, this research studied the fermentation conditions of exogenous non-starch polysaccharide degrading enzymes (NSPases) and probiotics for the mixed okara and CM. In this study, only evaluate the effect of solid-state fermentation on the reducing sugar content, lactic acid contents, butyric acid contents and soy globulins contents of okara and CM. However, lactic acid contents, butyric acid contents and soy globulins contents is not related to non-starch polysaccharides. Therefore, this paper should study the change of indexes which are related to non-starch polysaccharides, such as fiber content, polysaccharide content, monosaccharide content, etc.
Answer:
To investigate the NSP content, we had checked the neutral detergent fiber (NDF) and the result showed in page 8, line 291-296 and Figure 2 (line 297).
In Result section:
Page 7, line 291-296.
“Moreover, on NDF investigation (Figure 2c), this exogenous enzyme was revealed to reduce the amount of NDF in the NSP enzyme group compared to the CTRL group and significantly decreased NDF in the mix group compared to others (p < 0.05). The NDF of FOCM was further decreased when probiotics and exogenous NSP enzymes were added to SSF simultaneously (Figure 2c).”
- Figure
The title of figures can be simplified. For example, the title of Figure 1 can be changed to: Effect of different initial moisture content on Lactobacillus species (a) and C. butyricum (b) growth in SSF of okara and CM.
Answer:
Thank you for your suggestion. We have rewritten all the figure legends.
In Result section:
Page 7, line 278-281, Figure 1.
Figure 1. Effect of different initial moisture content on Lactobacillus species (a) and C. butyricum (b) growth in mixed SSF of okara and CM.
Page 7-8, line 298-300, Figure 2.
Figure 2. Effect of the NSPases supplementation on total Lactobacillus viable count (a); water contents (b); and neutral detergent fiber contents (c) of okara and CM fermentation under various fermentation product content.
Page 8, line 320-322, Figure 3.
Figure 3. Effect of 55% initial moisture of fermentation okara and CM with the combination of mixed probiotics and NSPases supplementation on reducing sugar content (a); lactic acid contents (b); and butyric acid contents (c) with different fermentation duration.
Page 13, line 418-423, Figure 6.
“Figure 6. Comparison of the bacteria communities of the ileal contents by advanced analysis. Principal component analysis (a) plots of basal diet as the control (CTRL), basal diets plus 1.25% of FOCM (FOCM1.25), basal diets plus 2.5% of FOCM (FOCM2.5), and basal diets plus 5.0% of FOCM (FOCM5.0) (n=3). Principal coordinate analysis of weighted UniFrac (b) and unweighted UniFrac (c) distance of the ileum bacterial communities from CTRL, FOCM1.25, FOCM2.5, and FOCM5.0 (n=3).”
Page 14, 425-428, Figure 7.
“Figure 7. Comparative analysis of the ileal contents across the samples. The beta diversity index of the ileal contents from the basal diet as control (CTRL), basal diets plus 1.25% of FOCM (FOCM1.25), basal diets plus 2.5% of FOCM (FOCM2.5), and basal diets plus 5.0% of FOCM (FOCM5.0) based on weighted UniFrac metrics (n=3) (a) and unweighted UniFrac metrics (n=3) (b).”
- Figure 2: It is really confusing about the group of probiotics, NSPases and mix. There is no introduction to these three groups in the material method.
Answer:
Thank you for your comment. To raise the explanation about it, we have added the information of each group in methods and figure legend of Figure 2 as follows:
In Methods section:
Page 4, line 140-145
“The experimental groups were divided into (1) mixed okara and copra meal (control, CTRL), (2) mixed okara and copra meal plus exogenous NSPases (NSP enzyme), (3) mixed okara and copra meal plus probiotics (Probiotics), (4) mixed okara and copra meal plus probiotics and NSPases (mixed). All experiments were incubated at 37°C and performed in triplicate. The FOCM were then dried at 50°C for 24 h, homogenized through mechanical agitation, and stored at 4°C prior to further analysis.”
In Figure 2:
Page 8, line 298-302.
“Figure 2. Effect of the NSPases supplementation on total Lactobacillus viable count (a); water contents (b); and neutral detergent fiber contents (c) of okara and CM fermentation under various fermentation product content. Mixed okara and copra meal (control, CTRL), mixed okara and copra meal plus exogenous NSPases (NSP enzyme), mixed okara and copra meal plus probiotics (Probiotics), mixed okara and copra meal plus probiotics and NSPases (mixed)”
In addition, Lactobacillus species and Clostridium butyricum were used as probiotics in this study. Why only Lactobacillus was detected, but not Clostridium butyricum?
Answer:
Thank you for your comment and question. We also tested the relative abundance of Lactobacillus species and Clostridium butyricum in FOCM (data not shown in the manuscript). From the heat map analysis (as below, 0 hr versus 60 hr fermentation), there was only the Lactobacillus species but no Clostridium butyricum in FOCM after 60 hr fermentation. This is very likely to occur, due to bacterial competition. In addition, bacterial secondary metabolites in the form of lactic acid are produced by lactic acid bacteria. Therefore, we only perform a total viable count test for lactic acid bacteria (Lactobacillus species).
Figure 1. Heatmap of species abundance of the microbiome from the FOCM.
Abundance distribution of dominant 35 genera (Y-axis) across all samples (X-axis) were displayed in the species abundance heatmap (n=3).
Author Response File: Author Response.pdf
Reviewer 3 Report
The manuscript was properly conducted and findings reported are important for poultry production and health. The paper contains important data health of chickens under different dietary treatments. The Authors investigated an interesting topic and the objective of the paper is of worldwide interest and fits well within the overall scope of the journal. Results were properly reported and the findings have been accurately discussed and compared with other recently published papers.
Author Response
Thank you for your helpful comments. We have revised our manuscript accordingly and feel that your comments helped clarify and improve our manuscript. Please find our response (in bold) to reviewer’s specific comments below.
Comments and Suggestions for Authors:
- The manuscript was properly conducted and findings reported are important for poultry production and health. The paper contains important data health of chickens under different dietary treatments. The Authors investigated an interesting topic and the objective of the paper is of worldwide interest and fits well within the overall scope of the journal. Results were properly reported and the findings have been accurately discussed and compared with other recently published papers.
Answer: Thank you for your very careful review of our paper.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Improvements have been made in response to the points raised. I wish you good luck in your research.
Reviewer 2 Report
This paper is acceptable for publication in its present form.
